1. Field of the Invention
The present invention relates generally to the field of agricultural biotechnology. More particularly, it concerns methods and compositions for the efficient identification of transgenic plant seeds.
2. Description of Related Art
Many attempts have been made to genetically engineer desired traits into plant genomes by introduction of exogenous genes using genetic engineering techniques. The uptake of new DNA by recipient plant cells has been accomplished by various means, including Agrobacterium infection (Nester et al., 1984), polyethylene glycol (PEG)-mediated DNA uptake (Lorz et al., 1985), electroporation of protoplasts (Fromm et al., 1986) and microprojectile bombardment (Klein et al., 1987).
An important aspect of the success achieved in transforming plants has been the ability to select or screen for transformed cells and/or plants. Most of the first successes in plant transformation relied on utilization of selectable markers for identification of transformed cells. Genes which have been used for selection of transformed plant cells include, for example, a neomycin phosphotransferase gene (Potrykus et al., 1985) which provides resistance to kanamycin, paromomycin and G418; a bar gene which codes for bialaphos or phosphinothricine resistance (U.S. Pat. No. 5,550,318); a mutant aroA gene which encodes an altered EPSP synthase protein conferring glyphosate resistance (Hinchee et al., 1988); a nitrilase gene such as bxn from Klebsiella ozaenae which confers resistance to bromoxynil (Stalker et al., 1988); a mutant acetolactate synthase gene (ALS) which confers resistance to imidazolinone, sulfonylurea or other ALS inhibiting chemicals (European Patent Application 154,204, 1985); a methotrexate resistant DHFR gene (Thillet et al., 1988); a dalapon dehalogenase gene that confers resistance to the herbicide dalapon; and a mutated anthranilate synthase gene that confers resistance to 5-methyl tryptophan.
More recently, interest has increased in utilization of screenable markers. One particularly useful screenable marker which has been discovered is the green fluorescent protein (GFP) (Sheen et al., 1995). GFP was first cloned and sequenced from the cnidarian Aequorea victoria (Prasher et al, 1992). Since then, it has been expressed in a variety of organisms, ranging from bacteria (Leff and Leff, 1996), fungi (Spellig et al., 1996), invertebrates (Plautz et al., 1996), vertebrates (Zolotukhin et al., 1996) and plants (Haseloff et al., 1997; Reichel et al., 1996; Sheen et al., 1995; Tian et al., 1997). An important advantage of GFP is that it can be assayed non-destructively. Firefly luciferase represents another useful fluorescent marker which can be assayed non-destructively, although detection requires addition of an exogenous substrate (luciferin) for detection (Ow et al, 1986; Millar et al, 1995). In contrast, the assay of the screenable marker GUS is cytotoxic, and therefore, recovery of live transformed cells is difficult (Jefferson et al., 1987).
One means that has been employed for the utilization of screenable markers, such as GFP, has been the fusion of the screenable marker with a selectable marker. Fusion proteins have been made, for example, between GFP and the selectable marker neomycin phosphotransferase (NPTII) (Genbank Accession No. AF004665), between GFP and hygromycin phosphotransferase (Lybarger et al. 1996), between the firefly luciferase gene (luc) and the neomycin phosphotransferase (NPTII) (Barnes, 1990), and between the ALS gene and GFP or GUS (WO 97/41228). The screening of transgenic maize cells based on GFP expression was described in WO 97/41228.
The identification of transgenic seeds using a GFP screenable marker also was described in WO 97/41228. In this case, however, the GFP gene was constitutively expressed using the ubiquitin promoter. It was found that seeds expressing GFP did not germinate when planted in soil, and could only be germinated by excision of embryos and plating on growth medium.
The failure to identify a method of non-destructively identifying transgenic seeds which can be directly germinated has represented a significant hindrance to breeders during the development of novel transgenic plant lines. Without labor-intensive embryo-rescue, current technology requires the growing and assaying of potentially transgenic seeds. The identification of a direct means for selection of transgenic cells, regeneration of plants from the transgenic cells and the screening of transgenic seeds which could, in turn, be directly advanced in breeding protocols would greatly improve scientists abilities to develop novel transgenic plant lines for the benefit of consumers.
The current invention seeks to overcome deficiencies in the prior art by providing methods and composition for the efficient identification of transgenic seeds. Therefore, in one aspect, the current invention provides a construct comprising a screenable marker gene operably linked to an aleurone-specific promoter. This screenable marker gene can be provided as a gene fusion between the screenable marker gene and a selectable marker gene, wherein the gene fusion is operably linked to an aleurone-specific promoter. In particular embodiments of the invention, the screenable marker gene is selected from the group consisting of a GFP gene, a luciferase gene, and an R gene. In other embodiments of the invention, the selectable marker gene comprises a gene selected from the group consisting of NPTII, bar, EPSPS, anthranalite synthase and dalapon dehalogenase. In still other embodiments of the invention, the aleurone-specific promoter is selected from the group consisting of an oleosin promoter, globulin 1 promoter, a barley LTP2 promoter, alpha-amylase promoter, chitinase promoter, beta-glucanase promoter, cysteine proteinase promoter, glutaredoxin promoter, HVA1 promoter, serine carboxypeptidaseII promoter, catalase promoter, alpha-glucosidase promoter, beta-amylase promoter, VP1 promoter, and bronze2 promoter. The oleosin promoter may be an L3 oleosin promoter. The marker gene constructs of the invention may additionally comprise first, second, third, or any additional number of exogenous genes which can physically be placed on the construct. The exogenous gene may be operably linked to a second promoter.
In another aspect, the invention provides a transgenic plant comprising a screenable marker gene operably linked to an aleurone-specific promoter. This screenable marker gene can be provided as a gene fusion between a selectable marker gene and the screenable marker gene, wherein the gene fusion is operably linked to an aleurone-specific promoter. In one embodiment of the invention, the screenable marker gene is selected from the group consisting of a GFP gene, a luciferase gene, and an R gene. In other embodiments of the invention, the screenable marker is a GFP gene. The selectable marker gene may comprise any suitable gene, for example, bar, EPSPS, NPTII, anthranalite synthase or dalapon dehalogenase. The promoter may be selected from the group consisting of an L3 oleosin promoter, a globulin 1 promoter, and a barley LTP2 promoter. The plant may be a monocotyledonous plant, and may be further defined as a plant selected from the group consisting of maize, rice, wheat, barley, oat, rye, millet, sorghum, sugarcane and turfgrass. In particular embodiments of the invention, the monocotyledonous plant is maize. The plant may also be a dicotyledonous plant, and may still further be selected from the group consisting of cotton, soybean, tomato, potato, citrus, and tobacco. In particular embodiments of the invention, the dicotyledonous plant is soybean.
In still yet another aspect, the invention provides a progeny plant of any generation of a transgenic plant comprising a screenable marker gene operably linked to an aleurone-specific promoter, wherein the progeny comprises said screenable marker gene. This screenable marker gene can be provided as a gene fusion between a selectable marker gene and the screenable marker gene, wherein the gene fusion is operably linked to an aleurone-specific promoter.
In still yet another aspect, the invention provides a method of identifying transgenic seeds comprising the steps of: (a) obtaining a transgenic plant, the cells of which comprise a screenable marker gene operably linked to an aleurone-specific promoter; (b) cultivating said plant until seed set; (c) collecting seeds produced on said plant; and (d) screening said seeds based on a phenotype conferred upon seeds by said screenable marker gene. In one embodiment of the invention, the screenable marker gene is provided as a gene fusion between a selectable marker gene and the screenable marker gene, wherein the gene fusion is operably linked to an aleurone-specific promoter. In particular embodiments of the invention, the screenable marker gene is selected from the group consisting of a GFP gene, a luciferase gene, and an R gene. The transgenic plant may be a monocotyledonous plant, and further, may be selected from the group consisting of maize, rice, wheat, barley, oat, rye, millet, sorghum, sugarcane and turfgrass. In particular embodiments of the invention, the monocotyledonous plant is maize. The transgenic plant may also be a dicotyledonous plant, and further defined as selected from the group consisting of cotton, soybean, tomato, potato, citrus, and tobacco. In particular embodiments of the invention, the dicotyledonous plant is soybean.
In the method of identifying seeds provided by the invention, the transgenic plant may further comprise an exogenous gene encoding a selected trait, wherein the exogenous gene is genetically linked to said screenable marker gene. The exogenous gene may still further comprise a promoter and 3xe2x80x2 region operably linked to said exogenous gene. In particular embodiments of the invention, the exogenous gene encodes a trait selected from the group consisting of an insect resistance gene, a viral disease resistance gene, a bacterial disease resistance gene, a fungal disease resistance gene, a nematode disease resistance gene, a herbicide resistance gene, a gene affecting grain composition or quality, a nutrient utilization gene, a mycotoxin reduction gene, a male sterility gene, a selectable marker gene, a screenable marker gene, a negative selectable marker, a gene affecting plant agronomic characteristics, and an environment or stress resistance gene. The screenable marker gene may also comprise a 3xe2x80x2 region operatively linked to the gene, and also an element enhancing the expression of said gene. In particular embodiments of the invention, the screening of collected seeds is automated.
In still yet another aspect, the invention provides a method of plant breeding comprising the steps of: (a) obtaining first and second plants, wherein cells of one or both of said first or second plants comprises a screenable marker gene operably linked to an aleurone-specific promoter; (b) crossing said first and second plants; and (c) collecting the seeds resulting from said crossing. The method may further comprise the step of: (d) selecting transgenic seeds from said collected seeds based on a phenotype conferred upon said transgenic seeds by said screenable marker gene. The screenable marker gene can be provided as a gene fusion between a selectable marker gene and the screenable marker gene, wherein the gene fusion is operably linked to an aleurone-specific promoter. In particular embodiments of the invention, the screenable marker gene may be selected from the group consisting of a GFP gene, a luciferase gene, and an R gene. In other embodiments of the invention, the selectable marker gene is selected from the group consisting of bar, EPSPS, NPTII, anthranalite synthase and dalapon dehalogenase. In still further embodiments of the invention, the aleurone-specific promoter is selected from the group consisting of an oleosin promoter, globulin 1 promoter, barley LTP2 promoter, alpha-amylase promoter, chitinase promoter, beta-glucanase promoter, cysteine proteinase promoter, glutaredoxin promoter, HVA1 promoter, serine carboxypeptidaseII promoter, catalase promoter, alpha-glucosidase promoter, beta-amylase promoter, VP1 promoter, and bronze2 promoter. Where the promoter is an oleosin promoter, an L3 oleosin promoter may be used.
In the method of plant breeding, the first and second transgenic plants may be monocotyledonous plants, and may be selected from the group consisting of maize, rice, wheat, barley, oat, rye, millet, sorghum, sugarcane and turfgrass. In particular embodiments of the invention, the monocotyledonous plants are maize plants. The plants may also be dicotyledonous plants, and can be selected from the group consisting of cotton, soybean, tomato, potato, citrus, and tobacco. In one embodiment of the invention, the dicotyledonous plants are soybean plants. In other embodiments of the invention, the step of selecting can be automated.
In still yet another aspect, the invention provides a method of plant breeding comprising the steps of: (a) obtaining a transgenic plant, the cells of which comprise a screenable marker gene operably linked to an aleurone-specific promoter; (b) allowing said plant to self fertilize; and (c) collecting the seeds produced on said plant. The method may further comprise the step of: (d) selecting transgenic seeds from said collected seeds based on the phenotype conferred upon said transgenic seeds by said screenable marker gene. The screenable marker gene can be provided as a gene fusion between a selectable marker gene and the screenable marker gene, wherein the gene fusion is operably linked to an aleurone-specific promoter. In particular embodiments of the invention, the screenable marker gene is selected from the group consisting of a GFP gene, a luciferase gene, and an R gene. In other embodiments of the invention, the selectable marker gene is selected from the group consisting of bar, EPSPS, NPTII, anthranalite synthase and dalapon dehalogenase. In still further embodiments of the invention, the aleurone-specific promoter is selected from the group consisting of an oleosin promoter, globulin 1 promoter, a barley LTP2 promoter, alpha-amylase promoter, chitinase promoter, beta-glucanase promoter, cysteine proteinase promoter, glutaredoxin promoter, HVA 1 promoter, serine carboxypeptidaseII promoter, catalase promoter, alpha-glucosidase promoter, beta-amylase promoter, VP1 promoter, and bronze2 promoter. In certain embodiments of the invention, the oleosin promoter may be an L3 oleosin promoter. comprises a globulin 1 promoter. The transgenic plant may be a monocotyledonous plant, and may still further be selected from the group consisting of maize, rice, wheat, barley, oat, rye, millet, sorghum, sugarcane and turfgrass. In particular embodiments of the invention, the monocotyledonous plant is a maize plant. The transgenic plant may also be a dicotyledonous plant, and may further be selected from the group consisting of cotton, soybean, tomato, potato, citrus, and tobacco. In particular embodiments of the invention, the dicotyledonous plant is a soybean plant. In other embodiments of the invention, the step of selecting may be automated.